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An RNA-Centric Global View of Clostridioides Difficile Reveals Broad 2 Activity of Hfq in a Clinically Important Gram-Positive Bacterium

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An RNA-Centric Global View of Clostridioides Difficile Reveals Broad 2 Activity of Hfq in a Clinically Important Gram-Positive Bacterium bioRxiv preprint doi: https://doi.org/10.1101/2020.08.10.244764; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 An RNA-centric global view of Clostridioides difficile reveals broad 2 activity of Hfq in a clinically important Gram-positive bacterium 3 Manuela Fuchs 1§, Vanessa Lamm-Schmidt 1§, Falk Ponath 2, Laura Jenniches 2, Lars Barquist 2,3, 4 Jörg Vogel 1,2, Franziska Faber 1, * 5 6 1 Institute for Molecular Infection Biology (IMIB), Faculty of Medicine, University of Würzburg, D- 7 97080 Würzburg, Germany 8 2 Helmholtz Institute for RNA-based Infection Research (HIRI), D-97080 Würzburg, Germany 9 3 Faculty of Medicine, University of Würzburg, D-97080 Würzburg, Germany 10 § authors contributed equally 11 * Correspondence: 12 Franziska Faber: phone +49-931-3186280; email: [email protected] 13 14 Running title: primary transcriptome of Clostridioides difficile 15 Keywords: Clostridioides difficile, Transcriptional start site, Transcriptional termination site, 16 differential RNA-seq, noncoding RNA, antisense RNA, small RNA, post-transcriptional control, 17 Hfq bioRxiv preprint doi: https://doi.org/10.1101/2020.08.10.244764; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 18 ABSTRACT 19 The Gram-positive human pathogen Clostridioides difficile has emerged as the leading cause of 20 antibiotic-associated diarrhea. Despite growing evidence for a role of Hfq in RNA-based gene 21 regulation in C. difficile, little is known about the bacterium’s transcriptome architecture and 22 mechanisms of post-transcriptional control. Here, we have applied a suite of RNA-centric 23 techniques, including transcription start site mapping, transcription termination mapping and 24 Hfq RIP-seq, to generate a single-nucleotide resolution RNA map of C. difficile 630. Our 25 transcriptome annotation provides information about 5’ and 3’ untranslated regions, operon 26 structures and non-coding regulators, including 42 sRNAs. These transcriptome data are 27 accessible via an open-access browser called ‘Clost-Base’. Our results indicate functionality of 28 many conserved riboswitches and predict novel cis-regulatory elements upstream of MDR-type 29 ABC transporters and transcriptional regulators. Recent studies have revealed a role of sRNA- 30 based regulation in several Gram-positive bacteria but their involvement with the RNA-binding 31 protein Hfq remains controversial. Here, sequencing the RNA ligands of Hfq reveals in vivo 32 association of many sRNAs along with hundreds of potential target mRNAs in C. difficile providing 33 evidence for a global role of Hfq in post-transcriptional regulation in a Gram-positive bacterium. 34 Through integration of Hfq-bound transcripts and computational approaches we predict 35 regulated target mRNAs for the novel sRNA AtcS encoding several adhesins and the conserved 36 oligopeptide transporter oppB that influences sporulation initiation in C. difficile. Overall, these 37 findings provide a potential mechanistic explanation for increased biofilm formation and 38 sporulation in an hfq deletion strain and lay the foundation for understanding clostridial ribo- 39 regulation with implications for the infection process. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.10.244764; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 40 INTRODUCTION 41 Antibiotic-resistant bacteria are one of the major global threats to human health, endangering our 42 ability to perform a range of modern medical interventions. The obligate anaerobe, spore-forming 43 Clostridioides difficile (C. difficile) has become the leading cause of antibiotic-associated diarrhea 44 over the past two decades (Rupnik et al., 2009). In addition, C. difficile shows increasing numbers 45 of multi-resistant clinical isolates (Peng et al., 2017) and recurrent infections after antibiotic 46 therapy, which often leaves fecal microbiota transfer as the only clinical option (Khanna and 47 Gerding, 2019). 48 The clinical challenges posed by C. difficile have prompted much effort to understand how 49 this pathogen regulates virulence in response to environmental conditions. As a result, there is a 50 comprehensive body of literature focusing on toxin production and sporulation control by several 51 global metabolic regulators including CcpA, CodY, Rex and PrdR (Bouillaut et al., 2015; Martin- 52 Verstraete et al., 2016). Furthermore, several specialized and general sigma factors including 53 TcdR (Martin-Verstraete et al., 2016), Spo0A (Pettit et al., 2014), SigD (El Meouche et al., 2013; 54 McKee et al., 2013), SigH (Saujet et al., 2011) and SigB (Kint et al., 2017) have been linked to 55 virulence and metabolism, although the exact molecular mechanisms often remain unknown. 56 Importantly, most of this knowledge has been accumulated through detailed studies of individual 57 genes and promoters. By contrast, RNA-seq based annotations of the global transcriptome 58 architecture, which have accelerated research in the Gram-positive pathogens Listeria (Wurtzel 59 et al., 2012), Staphylococcus (Mader et al., 2016) and Streptococcus (Warrier et al., 2018), have not 60 been available for C. difficile so far. 61 This paucity of global knowledge about RNA output in C. difficile readily extends to post- 62 transcriptional control of gene expression. The bacterium is of particular scientific interest, being 63 the only Gram-positive species thus far in which deletion of hfq seems to have a large impact on 64 gene expression and bacterial physiology (Boudry et al., 2014; Caillet et al., 2014). Specifically, 65 deletion of hfq increases sporulation (Maikova et al., 2019), a crucial pathogenic feature of this 66 bacterium that enables transmission between hosts. In Gram-negative bacteria, Hfq commonly 67 exerts global post-transcriptional control by facilitating short base pairing interactions of small 68 regulatory RNAs (sRNAs) with trans-encoded target mRNAs (Holmqvist and Vogel, 2018; Kavita 69 et al., 2018) but its role in Gram-positive bacteria remains somewhat controversial. For example, 70 recent work on the function of Hfq in Bacillus subtilis (B. subtilis), the model bacterium for 71 Firmicutes, revealed in vivo association with a subset of sRNA (Dambach et al., 2013), but 72 comparative analyses of a wild-type and hfq knockout strain revealed only moderate effects on 73 sRNA and mRNA transcript levels (Hammerle et al., 2014) and the absence of any significant 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.10.244764; this version posted August 10, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 74 growth defect (Rochat et al., 2015) which lead to the conclusion that Hfq plays a minor role in 75 post-transcriptional regulation in B. subtilis. 76 Previous efforts using in silico methods (Chen et al., 2011) and RNA-sequencing 77 (Soutourina et al., 2013) have predicted >100 sRNA candidates in C. difficile, which would suggest 78 the existence of a large post-transcriptional network. However, under which conditions these 79 sRNAs are expressed, which targets they regulate, and whether they depend on Hfq remain 80 fundamental open questions. 81 Another important feature of post-transcriptional control in C. difficile are cis-regulatory 82 RNA elements. There has been pioneering work deciphering the function of genetic switches 83 located in the 5’ untranslated region (5’ UTR) of the flgB operon, containing the early stage 84 flagellar genes, and the cell wall protein encoding gene cwpV (Anjuwon-Foster and Tamayo, 2017; 85 Emerson et al., 2009). Moreover, cyclic di-GMP responsive riboswitches were shown to regulate 86 biofilm formation and toxin production (Bordeleau et al., 2011; McKee et al., 2018; McKee et al., 87 2013; Peltier et al., 2015). However, other members of the many different riboswitch classes or 88 common cis-regulatory elements such as RNA thermometers have not been systematically 89 searched for. Combined with the nascent stage of sRNA biology in C. difficile, this argues that global 90 approaches are needed to understand the full scope of post-transcriptional regulation in this 91 important human pathogen. 92 In the present study, we have applied recently developed methods of bacterial RNA 93 biology (Hor et al., 2018) to construct a global atlas of transcriptional and post-transcriptional 94 control in C. difficile. Our approach demonstrates how integration of different RNA-sequencing 95 based methods readily reveals new regulatory functions. Accordingly, we were able to predict 96 regulatory interactions between a newly annotated Hfq-binding sRNA and target mRNA 97 candidates. Overall, we provide evidence for extensive Hfq-dependent post-transcriptional
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